Soil test

ABSTRACT

A test for determining the bioavailable fraction of an organic pollutant present in soil comprises determining the fraction of the organic pollutant present in the soil which may be extracted by a cyclodextrin or derivative thereof.

The present invention relates to a test for the analysis of soil, sand,clay and other earth materials (all collectively referred to herein as“soil” for convenience) contaminated with persistent organic pollutantsto determine the bioavailable fraction of the pollutant in the soil,particularly but not exclusively for the purposes of deciding whether ornot the soil may be purified using a bioremediation strategy.

Various industrial processes may result in contamination of soil withpersistent organic pollutants, e.g. polycyclic aromatic hydrocarbons.Examples of industrial processes resulting in such pollution includeincineration processes, wood treatment sites, gas works and powerstations. It is obviously desirable, for environmental reasons, thatsuch soil be purified. One such strategy for purification isbioremediation in which the soil may be treated by composting,air-sparging or bioaugmentation or by means of a pump and treatprocedure or by using a bioreactor so that organisms proliferating inthe soil are able to “digest” the pollutants. However the success orotherwise of a bioremediation strategy depends on the bioavailability ofthe pollutants in the soil. In other words, only a certain fraction(i.e. the bioavailable fraction) of the pollutants in the soil will beremoved by the bioremediation strategy. If this fraction is relativelylow then the bioremediation technique, whilst reducing the total levelof organic pollutants, may not necessary bring the level down below thatrequired by legislation.

The bioavailability of a persistent organic pollutant in soil isdependent on a number of factors. For example, it has been observedthat, as the length of time an organic compound remains in contact withsoil increases, the ability for that compound to be degraded bymicro-organisms decreases (Hatzinger and Alexander, 1995). The rate atwhich degradation occurs also decreases (Hatzinger and Alexander, 1995).Similarly, a decrease in solvent extractability has been observed ascompound/soil contact time increases (Hatzinger and Alexander, 1995;Kelsy et al, 1997). This decrease in compound availability, with time,has been termed “ageing”. It is the rapidly desorbed fraction whichequates to the bioavailable fraction (Cornelissen 1998).

The nature and extent of ageing is dependent upon soil structure,intra-soil processes and the compound's intrinsic properties.Fundamental parameters include (Brusscau et al. 1991; Jones et al,1996): aqueous solubility, vapour pressure and octaonol: water partitioncoefficient (Kow). It has been proposed that ageing is the amalgamationof a number of intra-soil process including: sorption onto soilparticles (Ball and Roberts, 1991a; Fu et al, 1994; Burgos et al, 1996),diffusion into spatially remote areas such as soil macro and micro pores(Ball and Roberts, 1991b; Beck et al, 1995, Burgos et al. 1996;Pignatello and Xing, 1996) and the entrapment within soil organic matter(Brusseau et al, 1991; Fchen et al; Fu et al, 1994). Neutral organiccompounds can interact with the soil through a number of attractiveforces, such as, dipole-dipole, dipole-induced dipole and hydrogenbonding (Pignatello and Xing, 1996).

Observation indicates that compound sorption to soil exhibits a biphasicbehaviour i.e. following an initial rapid phase of sorption there is asubsequent slower but more prolonged period of sorption (Jones et al,1996; Pignatello and Xing, 1996). This biphasic behaviour has also beenobserved in thermal desorption studies (Fu et al, 1994), interestinglysorption/desorption processes have a distinct hysterics, where morecompound is sorbed than can subsequently be desorbed (Pignatello, 1991:Fu et al, 1994).

Intra-particle diffusion of compounds into macro- and micro-pores mayimpede biodegradation on the grounds that pores are of a size smallenough to exclude micro-organisms (Chung et al, 1993). Exclusion ofmicro-organisms on the grounds of size is also postulated to occur wherecompounds becomes entrapped within large humic macromolecules (Fu et al,1994). It has been noted that where biodegradation has stopped in thecase of aged materials, pulverisation (using a ball mill) of the soilresulted in continued biodegradation (Steinberg et al, 1987). Thissupports the hypothesis that compounds become trapped within soilmicropores as aging proceeds. Investigation (Wu and Gschwend, 1986) intothe influence of particle size on the rate of sorption/entrapment,revealed much greater rates of uptake for small particles when comparedto large ones.

It is evident that the fate and behaviour of organic compounds withinthe soil environment is dependent on a complex array of processes.Processes which ultimately govern bioavailability, and thereby dictatethe feasibility of bioremediation strategies. At one extreme, wherecompounds are completely available remediation by a biotechnology shouldprove favourable. At the other extreme where compounds are recalcitrantin nature, such a strategy may fail,

There is a need for a test for the analysis of soil contaminated withpersistent organic pollutants to determine whether organic compounds arebioavailable and thereby whether or not a bioremediation strategy willbe capable of effecting a sufficient reduction (e.g. to meet legislativerequirements) of the amount of pollutants in the soil.

EP-A-0 613 735 discloses a method for the extraction of organicpollutants from contaminated soils. The method involves treatment of thesoil with an aqueous solution of a cyclodextrin or derivative thereof.It is stated in this prior specification that cyclodextrins (or theirderivatives) enhance the desorption of contaminants from the soil andthat this activity can be utilised for the enhancement or the rate ofbioremediation of soils contaminated with organic pollutants. As such,the prior specification proposes that the bioremediation of soilscontaminated with organic pollutants may be enhanced by treatment of thesoil with a cyclodextrin (or derivative thereof) to increase thebioavailability of contaminants to micro-organisms which are capable ofdegrading the contaminants. However, we have discovered (and this formsthe basis of the present invention) that the amount of persistentorganic pollutant which a cyclodextrin (or derivative) is able toextract from a soil sample is representative of the amount of thepollutant which is bioavailable and which may therefore be removed by abioremediation strategy.

On this basis, the present invention provides a test for determining thebioavailable fraction of an organic pollutant present in soil comprisingdetermining the fraction of the organic pollutant present in the soilwhich may be extracted by a cyclodextrin or derivative thereof.

The present invention is thus to be distinguished from the disclosure inEP-A-0 613 735 in a fundamental way. In the prior specification, thebioremediation strategy is effected by treating the soil inter alia witha cyclodextrin purportedly to increase the bioavailability of theorganic pollutant. In contrast, the present invention provides not abioremediation procedure per se but rather an analysis procedure inwhich an extraction (of a soil containing persistent organic pollutants)is effected using a cyclodextrin (or derivative thereof) to determinethat fraction of the total pollutant which is bioavailable. To a firstapproximation, the fraction so determined enables a simple calculationto be performed to determine the level of pollutant which would remainin the soil after the bioremediation treatment. Thus it is possible todetermine whether the soil is suitable for treatment by bioremediatione.g. to ensure that the remaining pollutant is below legislative maxima.

A preferred test procedure in accordance with the invention comprisesthe steps of

(a) analysing a first sample of the soil to determine the total amount(or representation thereof) of the organic pollutant which it is desiredto remove by a bioremediation strategy,

(b) extracting a sample of the soil with a cyclodextrin (or derivativethereof) to determine the maximum amount (or representation thereof) ofthe pollutant in this sample which can be extracted by the cyclodextrin(or derivative thereof, and

(c) using the results from (a) and (b) to determine the maximum fraction(or representation thereof) of the total pollutant which can beextracted by the cyclodextrin.

Cyclodextrins as used in the method of the invention are “bucket-shaped”molecules comprising a taurus of α-1, 4linked glucose units, with sixunits being denoted α-cyclodextrin, seven units being denotedβ-cyclodextrin and eight units being denoted γ-cyclodextrin. Theprocedure of the invention may use α-, β- and/or γ-cyclodextrin. It isalso possible to use a cyclodextrin derivative, e.g. analkyl-hydroxyalkyl or acyl derivative or any of the other derivativesdisclosed in EP-A-0 613 735. It is particularly preferred to use ahydroxy(C₁₋₄alkyl) derivative most preferablyhydroxypropyl-βcyclodextrin (HPCD) which has greater water solubilitythan β-cyclodextrin.

The method of the invention is applicable particularly, but notexclusively, to pollutants such as polyaromatic hydrocarbons,polychlorinated phenols and biphenyls, dioxines and furanes. If thepollutant is a polyaromatic hydrocarbon having two or three rings thenit is preferred that the cyclodextrin is a β-cyclodextrin. If thepollutant is a polyaromatic hydrocarbon with a greater number of ringsthen it is preferred that the cyclodextrin is a gamma-cyclodextrin.

Cyclodextrin is unstable at a pH below ca5. As such, it should beensured that extraction procedures utilising cyclodextrin are effectedat a higher pH. It is preferred that an unbuffered extraction be usedwhere feasable, i.e. provided that the soil pH is not sufficientlyacidic to dissociated the cyclodextrin macromolecule during extraction.Alternatively, although less preferred, a buffered solution may be usedto stabilize the cyclodextrin macromolecule. By way of example, thecyclodextrin may be dissolved in:

(i) a buffer comprising K₂HPO₄ (0.2 M) and citric acid (0.1 M) in aratio of 1:1.714 to provide a pH of 6;

(ii) a buffer comprising K₂HPO₄ (0.2 M) and KH₂PO₄ (0.2 M) in a ratio of1:0.639 to provide a pH of 7 or in a ratio of 1:0.056 to provide a pH of8; or

(iii) a buffer comprising Na₂CO₃ (0.1 M) and NaHCO₃ (0.1 M) in a ratioof 1:9 to provide a pH of 9.2.

The extraction with the cyclodextrin (or derivative thereof) must besuch that there is extracted from the soil the maximum amount ofpollutant which the cyclodextrin (or derivative) is capable ofextracting since otherwise the fraction of bioavailable pollutant in thesoil will be underestimated. This will generally imply that theextraction is effected with an excess of the cyclodextrin (orderivative). Thus, it may be necessary to extract two or more soilsamples with differing amounts of cyclodextrin (or derivative thereof)to ensure that the maximum amount of pollutant has been extracted. Thus,for example, a series of soil samples may be extracted withprogressively increasing amounts of cyclodextrin until a constant amountof pollutant is extracted, this being the maximum amount which may beextracted by means of the cyclodextrin.

Typically the concentration of the cyclodextrin in the solution to beused for extraction of the soil will be in the range 20 mM to 80 mMalthough we do not preclude values outside this range.

In order to determine whether or not a particular soil sample may beeffectively treated using a bioremediation strategy, it is necessary tocompare the maximum amount of pollutant which may be extracted by thecyclodextrin with the total amount of pollutant in the soil sample.Various procedures may be used for determining the total amount ofpollutants from the soil sample and various possibilities are detailedas (1)-(5) below.

(1) A convenient technique is exhaustive solvent extraction. A solventextract from the soil may be obtained by a Soxhlet procedure usingreflux extraction (cycling solvent vapour through the sample) e.g. for atotal of 8-12 hours. The solvent may, for example, be dichloromethane(although this tends to give an extract which is difficult to “clean-up”for the purposes of subsequent analysis), hexane (which is useful if thesoil sample is dry since it gives high extraction efficiency and cleanersamples), and a hexane:acetone mix.

(2) A Saponification extraction may be used involving reflux of amixture of the soil and a saponification lye comprised of 2M KOH mixedwith methanol in a ratio 1:4. Reflux may be effected for 6 hours, afterwhich the heat source is removed, the system allowed to cool, and thecondensers back washed with fresh saponification lye.

This is a harsher technique than (1) which actively breaks up the soilorganic matter thereby releasing very tightly bound compounds.

(3) A sonication technique may be used in which the soil sample to beextracted is mixed with solvent and then placed in a sonicating bathwhere it is subjected to sonic waves (as an alternative to refluxing in(1) and (2).

(4) The soil sample to be extracted may be mixed with solvent and thensubjected to microwave radiation. This provides a rapid means ofobtaining extracts.

The bulk extracts obtained in accordance with procedures (1)-(4) abovemust generally be concentrated (e.g. by rotary evaporation) to removeexcess solvent but not the compounds of interest. The concentratedextracts may then be fractionated to give a clean fraction containingthe compounds of interest. Fractionation may most conveniently beachieved by use of chromatographic columns either of a conventionalnature, e.g. comprising silica or alumina, or by for example gelpermeation chromatography. If necessary, the resultant purified andcondensed (?) sample may be reduced in volume again and this may beachieved on a heating block under a stream of nitrogen. The samples arethen ready for analysis by, for example, gas chromatography/massspectrometry or high performance liquid chromatography.

(5) A Super Critical Fluid Extraction may be employed and involvespurging the soil sample of interest with liquid carbon dioxide torelease the sample compounds of interest. This technique produces cleansamples which are virtually ready for analysis but can only be appliedto volatile and semi-volatile pollutants.

(6) Volatile organic compounds can also be extracted from soil by apurge and trap method.

It will be appreciated from the above that it can be difficult todetermine the actual, absolute total amount of pollutant in the soiland, in fact, different types of pollutants may require differentextraction procedures. For this reason, it is convenient to define themaximum amount of a particular pollutant present in a soil by referenceto a standard extraction procedure. In accordance with the invention, itis preferred that the procedure for determining the total amount ofvolatile organic pollutants present in the soil is Method US EPA 8240Bas disclosed, for example, in “Guidance Manual on Sampling, Analysis,and Data Management for Contaminated Sites, Volume II: Analytical MethodSummaries” as published by the Canadian Council of Ministers of theEnvironment (the National Contaminated Sites Remediation Program) or isan extraction procedure which is at least as efficient as thisprocedure. A volatile organic pollutant may be defined as one having aboiling point of less than 200° C. or may be defined by the compound'sHenry's Law constant. Volatile organic compounds have relatively highHenry's Law constants, i.e.

10³-10⁵ Pa m³ mol⁻¹ for hydrocarbons

10-10⁵ Pa m³ mol⁻¹ for halogenated hydrocarbons

0.1-100 Pa m³ mol⁻¹ for ethers

(see Mackay et al. 1993).

For determining the total amount of semi-volatile organic pollutants itis preferred that the procedure is US EPA 8270B as disclosed in theaforementioned reference, or as an extraction procedure which is atleast as efficient as this procedure. A semi-volatile organic compoundmay be defined as one having a boiling point of at least 200° C. or maybe alternatively defined as a compound having a LOG octanol-airpartition coefficient (K_(QA) greater than 4 where K_(QA) is defined (atequilibrium) as$K_{OA} = \frac{\left\lbrack {{Organic}\quad {Pollutant}} \right\rbrack_{Octanol}}{\left\lbrack {{Organic}\quad {Pollutant}} \right\rbrack_{Water}}$

It must however be appropriate that any one country may have prescribedtests for determining the total amount of persistent organic pollutantspresent in soil and for any particular to which this patent applicationextends the term “total amount of pollutant in the soil sample” is to beunderstood as the amount which can be extracted by the prescribed test.

It will be appreciated from the foregoing description that with theknowledge of (i) the total pollutant content of the soil, and (ii) themaximum pollutant content which may be extracted by the cyclodextrin itis possible to determine for the soil sample the fraction of pollutantwhich may be removed by a bioremediation strategy. It is thus possibleto make a decision as to whether such a strategy would be effected fortreating the soil, e.g. to bring the level of pollutants belowlegislative limits.

Although the present invention has particular application in determiningwhether or not soil is suitable for a bioremediation strategy, there areother aspects to the invention. In particular, it is possible to use thebioavailable fraction to make a decision as to the degree of hazard (orotherwise) posed by a pollutant in soil. If the bioavailable fraction isrelatively low then the pollutant is immobilised and the soil is likelyto be “low hazard”. Conversely, if the bioavailable fraction isrelatively high then the soil may pose a significant hazard.

The invention will be described by way of example only with reference tothe following non-limiting Examples and accompanying FIG. 1 of thedrawings.

EXAMPLE 1

HPCD Concentration

A sample was “spiked” with 10 mg kg⁻¹ ¹⁴C-9phenanthrene. Samples of thesoil (1.25 g) were then extracted for 20 hours (sufficient time forextraction of the rapidly i.e. non-kinetically restrained fraction ofcompounds to be exchanged—see Example 2) with increasing concentrationsolutions (25 ml) of HPCD in water. The amount of activity exchangedinto the HPCD solution was measured as was the total activity present.The results are shown in Table 1 in which the activity units are dpm g⁻¹soil (where dpm stands for disintegrations per minute).

TABLE 1 Activity HPCD Exchanged Total % Exchangeable Concentration intoHPCD Activity with HPCD (mM) Solution Present solution  0  86.96 3116.79 2.79  5 1186.25 3116.79 38.06 10 1429.98 3116.79 45.88 20 1792.473116.79 57.51 40 2232.56 3116.79 71.63 50 2005.03 3116.79 64.33 602173.65 3116.79 69.74

These results demonstrate that maximum extraction of the organicpollutant (phenanthrene) was obtained using an HPCD solution having aconcentration of 40 mM.

EXAMPLE 2

Extraction Time

The above extraction was repeated but using an HPCD solution having aconcentration of 50 mM. However the extraction was terminated after 3,6, 12, 18, 24 and 40 hours. The results are shown in Table 2 in whichthe activity units are dpm g³¹ ¹ soil (where dpm stands fordisintegration per minute).

TABLE 2 Extraction Activity Total % Exchangeable Time Exchanged intoActivity with HPCD (h) HPCD Solution Present solution  0   0.0 4384.8 0.0  3 2813.4 4384.8 64.2  6 3219.2 4384.8 73.4 12 3192.1 4384.8 72.818 3264.6 4384.8 74.5 24 3368.6 4384.8 76.8 40 3314.9 4384.8 75.6

These results demonstrate that the rapidly i.e. non-kineticallyrestrained fraction of organic pollutant (phenanthrene) was extractedafter an extraction time of 6 h. Thus the use of a 20 h extraction time(Example 1) is valid for assessment.

EXAMPLE 3

Correlation of HPCD Extractability with Biodegradability

Soil was “spiked” to concentrations of 25 and 50 mg kg⁻¹. The aboveextraction was repeated but using a HPCD solution having a concentrationof 50 mM. Soil samples were aged for 1, 42 and 84 d prior to extraction.An extraction time of 20 h was used as before (Example 1 and 2 ). Thebiodegradability of the soil associated ¹⁴C-9-phenanthrene was assessedusing respirometry. Respirometers consisted of Erlyn,eyer flasks (250mL), to which soil (10 g) and water (30 mL) were added. The flasks wereinnoculated (10⁷-10⁸ g⁻¹ soil) with a catabolocally active microbialculture. The sealed flasks were shaken and the carbon dioxide evolvedfrom the biodegration of the phenanthrene trapped in potassium hydroxide(1 M, 1 mL.) Mineralisation was measured until it platcauted (240 h).The results are shown in Table 3 and FIG. 1.

Original “Spiked” Concentration Concentration Phenanthrene Extractedinto Mineralized by Ageing Concentration HPCD Solution microbes time (d)(mg/kg⁻¹) (mg/kg⁻¹) (mg/kg⁻¹)  1 25 19.5 18.2 42 25 17.6 14.9 84 25 15.514.7  1 50 29.9 32.0 42 50 15.4 17.8 84 50  1.0  1.1

The results indicate that the extraction of soil associated phenantheneby an aqueous HPCD solution as described above provides good correlation(slope=1.041; 1^(0.2)=0.980) with the amount of phenanthrene which canbe biodegraded by catabolically active microorganisms. Furthermoreprediction can be made over a range of concentrations and after a rangeof ageing times.

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What is claimed is:
 1. A test for determining a bioavailable fraction of an organic pollutant present in soil comprising determining a total amount of an organic pollutant in the soil and determining a maximum fraction of the organic pollutant present in the soil which may be extracted by a solution of a cyclodextrin or derivative thereof.
 2. A test as claimed in claim 1 comprising the steps of (a) analyzing a sample of the soil to determine a total amount, or representation thereof, of the organic pollutant which can be extracted from the soil sample, (b) extracting a sample of the soil with a solution of a cyclodextrin, or derivative thereof, to determine a maximum amount, or representation thereof, of the pollutant in this sample which can be extracted by the cyclodextrin, or derivative thereof, and (c) using the results from (a) and (b) to determine the maximum fraction, or representation thereof, of the total pollutant which can be extracted by the cyclodextrin.
 3. A test as claimed in claim 2 wherein for a volatile organic pollutant the total amount of that pollutant in the soil is determined in accordance with US EPA 8240B or a procedure which provides at least the same value of the total amount.
 4. A test as claimed in claim 2 wherein for a semi-volatile organic pollutant the total amount of that pollutant in the soil is determined in accordance with US EPA 8270B or a procedure which provides at least the same value of the total amount.
 5. A test as claimed in claim 1 wherein the extraction with the cyclodextrin or derivative thereof is effected using an unbuffered aqueous solution of the cyclodextrin or derivative thereof under conditions preventing dissociation of the cyclodextrin.
 6. A test as claimed in claim 1 wherein the extraction with the cyclodextrin or derivative thereof is effected using a buffered solution.
 7. A test as claimed in claim 6 wherein the buffer comprises H₂PO₄ ion containing salts and citric acid.
 8. A test as claimed in claim 7 wherein the salt is selected from the group consisting of the potassium salt and the sodium salt.
 9. A test as claimed in claim 6 wherein the buffer comprises HPO4²⁻ and H₂PO₄ ⁻ ion containing salts.
 10. A test as claimed in claim 9 wherein the salt is selected from the group consisting of the potassium salt and sodium salt.
 11. A test as claimed in claim 6 wherein the buffer comprises CO₃ ²⁻ and HCO₃ ⁻ ion containing salts.
 12. A test as claimed in claim 11 wherein the salt is selected from the group consisting of the potassium salt and the sodium salt.
 13. A test as claimed in claim 1 wherein the cyclodextrin or derivative thereof is used as a solution having a concentration of 20 mM to 80 mM.
 14. A test as claimed in claim 1 wherein the extraction with the cyclodextrin or derivative thereof is effected with a hydroxy(C₁₋₄ alkyl) cyclodextrin derivative.
 15. A test as claimed in claim 14 wherein the derivative is hydroxypropyl-β-cyclodextrin.
 16. A test as claimed in claim 1 wherein the total amount of the organic pollutant is determined using exhaustive solvent extraction.
 17. A method for determining whether or not a soil contaminated with organic pollutants is suitable for purification by a bioremediation strategy, the test comprising the steps of (i) determining the bioavailable fraction of the organic pollutants in accordance with the test procedure of claim 1, and (ii) using the fraction from (i) to make a decision as to whether or not the soil is suitable for bioremediation.
 18. A method for determining soil hazard comprising the steps of (i) determining the bioavailable fraction of the organic pollutants in accordance with the test procedure of claim 1, and (ii) using the fraction from (i) to make a decision as to the hazard posed by the soil. 